AGRICULTURAL SCIENCES

MUSHROOM CULTIVATION
(AGA -453)
B.Sc. (Hons.), B.Sc. Integrated MBA

Chandigarh University

Gharuan (Mohali)
Practical – 3 Collection & Identification of Mushrooms
Aim of the Practical: The students will learn the techniques to collect mushrooms from the
wild, their identification and preparation of pure culture following isolation techniques.
The Equipments and Tools Required:
While going for mushroom collection, one must carry a GPS, digital camera, ice boxes, cutting
knives, blades, rubber gloves, scissors, paper bags, polythene bags, paper napkin, wax papers,
blotting paper pieces, field guide book on mushrooms, umbrella, torch, a notebook and pen,
collecting baskets, first aid box, loose wearing with a hat and hunter shoes.
A. Collection of wild mushrooms:
Different types of mushrooms appear in meadows, fields or forests just after the first
showers in the rainy season. The following points should be taken care while collecting wild
fungi:
1. As and when you see the mushroom, firstly take a photograph and then collect it by
digging down to the base. If it is possible, try to collect different stages of mushroom.
Observe fruiting body for change in its color and also record the species of trees/ locality
of mushroom; after collections arrange the fruiting bodies in a single layer at the bottom
of basket. Collected fungi should be handled as little as possible and not bruised or
crushed.
2. The second important thing is the appearance of a fruiting body i.e., general shape, size
and colour should be noted. In addition to this, with the help of hand lens try to observe
the presence of volva or pseudorhiza, gill attachments to stipe or presence of gills/ tubes/
teeths.
3. Attempt should be made to compare the morphological characters of collected samples
with the ones given in guide book or with google.
4. Do not touch the fruiting body and never try to find out its taste in a hurry. And always
wear gloves while picking the mushrooms as direct contact with mushrooms may cause
allergic reactions to some individuals.
5. Though fungi such as many polypores and hydnums do not suffer much from handling,
hence these should be wrapped in paper and packed more closely.
6. Along with the locality, collection date and other evanescent characters, such as a
distinctive smell, change of colour when gently touched or bruised and so on, should be
noted.
7. Observe the fruiting bodies under microscope for micrometery and also record the color
 of spore print (especially for any unknown gill- fungi). This is done by gently removing
the stem from cap, laying the cap, gills downwards on a sterile white paper/ petriplate
and leaving it for some hours or overnight. The spore- powder deposited gives the colour
of spores, which is important for identification purposes.
8. Since fleshy fungi cannot be preserved in their natural form and colour, students should
make coloured drawings or make presentations by clubbing the photographs of different
mushrooms, which will provide permanent records.
B. Identification:
Points to be observed for Identification of Fleshy fungi:
General Appearance: Size, whether growing solitary or in groups, texture, colour, any change
of colour with age or on drying, presence or absence of veils in the young stage, presence of
volva etc.
Cap: Its morphology, size, shape, colour, nature of surface (whether smooth, slimy, scaly or
fibrillose, impervious to water), kind of margin, whether easily separated from stem.
Stem: Size, shape (whether equal in thickness throughout or thickened above or below), colour,
nature of surface, presence of rings or volva, whether the flesh is continuous with that of the cap
or distinct (cartilaginous).
Tubes: Length, colour, shape of mouth, mode of attachment to stem.
Gills: Colour when young and later, texture, thickness, whether crowded or distant (spaced),
whether all of the same length or of different lengths and method of attachment to the stem.
Flesh: Thickness, colour, any change of colour or exudation of a milky or colored juice when
cut, smell etc.
Spores: For identification purposes, the microscopic characters of spores like colour and other
anatomical details are necessary. The properly dried specimens are filled in air tight polythene or
paper packets and labelled.
After description, neat sketch or drawing is to be prepared.
Note: Make detailed notes about the habitat (whether the mushroom is growing on dead or living
tree or on soil or compost or near water bodies or grass or roots of uprooted trees etc.), time of
collection, distinct smell of some mushrooms to attract insects, in addition to this some
mushrooms change their colors while we touch them. Try to keep all the collected samples
separately.
C. Preparation of media for raising of Pure culture:
The pure cultures are raised on a convenient culture medium which is generally in solidified
state due to the addition of Agar-agar, a sea weed. The composition of media and the methods of
preparation are as given below:
1. Potato - dextrose Agar medium ( PDA)
Peeled and sliced potato ---- 250g
Dextrose ---- 20g
Agar –agar powder ---- 20g
Water ---- 1000ml
About 250 gram potatoes are peeled, cut into small pieces, boiled in water for 25-30 minutes
and filtered through a muslin cloth. The volume of the extract is raised to 1000 ml with water
and boiled along with dextrose and agar-agar powder so as to get a thoroughly mixed solution.
Before adding agar agar, the pH of media is to be adjusted to 7.0. After adding agar powder,
the media is transferred to test tubes or narrow mouthed Erlenmeyer flasks (for pouring media
in Petri plates sterilized in an oven at 180ºC for two hours ) followed by their plugging with
non-absorbent cotton and sterilization in an autoclave.
2. Potato -dextrose Yeast Agar Medium ( PDYA )
Just like preparation of PDA, PDYA can be prepared by adding 2g Yeast extract in the solution
for selected fungi.
3. Malt Extract Agar medium (MEA)
Malt extract ---- 25 g
Agar- agar powder ---- 20 g
Distilled water ---- 1000 ml (pH—7.0)
Malt extract and agar are mixed in 1 litre water and boiled by continuously stirring with a
glass rod so as to avoid formation of clumps.
4. Compost Extract Agar medium (CEA)
Pasteurized compost ---- 150 g
Agar –agar powder ---- 20 g
Water ---- 1000 ml (pH ----7.0)
Compost is boiled in 1.5 to 2.0 litre water for few minutes till volume of the water is reduced to
half and after filtering through muslin cloth, the volume is again made to 1 litre and autolclaved
after mixing agar powder in it and filling in the test tubes.
5. Malt Peptone Grain Agar Medium (MPGA)
Malt extract ---- 20g
Rye or Wheat grains ---- 5g
Yeast (Optional) ---- 2g
Agar-agar powder ---- 20g
Peptone ---- 5g (pH -7.0)
Wheat or rye grains are boiled in water for 1-1.5 hours; the filterate is mixed with other
ingredients and continuously stirred while heating before filling and autoclaving.
D. Isolation techniques
Isolation techniques for getting pure cultures and their maintenance:
There are two methods to have a mushroom culture - the Spore Culture and Tissue Culture
technique.
1. Spore Culture
a) Spore Print : In order to get a spore print or collection of spores , the cap from a healthy,
disease free mushroom is removed , surface cleaned with a swab of cotton dipped in alcohol
and placed on a clean sterilized white paper or on clean glass plate or on surface of the clean
glass slides .The surface nearby should be thoroughly sterilized. To prevent air flow, place a
glass jar or clean glass or cup over the cap surface. Spores will fall on the white paper or slide
surface within 24-48 hours exactly like radial symmetry of the gills .The spore print on the
paper can be preserved for a longer time by cutting and folding it into two halves.
b) Spore transfer and germination: In order to get a pure culture , the scalpel is sterilized by
keeping it on a burning flame for 8-10 seconds till it becomes hot red , cool it by dipping in a
sterilized medium, scrap some spores from the spore print taken on a paper or glass slide and
transfer them by gently streaking on the agar medium aseptically. Minimum, three agar dishes
should be inoculated for each spore print and the culture developed after its incubation at
appropriate temperature is known as multispore culture.
2. Tissue Culture: A small bit from the pileal region is cut with the help of a sterilized blade or
scalpel, washed several times in sterilized distilled water and dried in a clean tissue paper before
inoculating aseptically on a Petri plate or tube containing suitable culture medium. The
inoculated Petri plates are incubated at 25 ± I C for 6-12 days and observed at different intervals
for the mycelial growth. All Petri plates / glass tubes showing contaminations should be
discarded and only the ones with pure growth should be retained for further use after
ascertaining the purity and true to type nature of the culture.
Sub-culturing:
The pure culture of edible mushroom, once established either through spore culture or tissue
culture technique, is maintained properly in cool atmosphere or a refrigerator. Sub-culturing is
done from time to time by aseptically transferring a small piece of growing pure culture along
with the culture medium on the test tube slants containing same or other suitable medium. The
pure culture of a mushroom can be used for preparing master cultures for large scale spawn
production on commercial scale. It will be discussed in the next lesson in detail.